Phage-bacteria relationships revealed by a metagenomic survey of the bovine rumen microbiome
Overview Within the last couple of years, many research groups have turned to metagenomic based approaches to examine the structure and make up of microbial communities within the digestive tracts of ruminants. This work has greatly expanded our understanding of how different sp ecies occupy distinct metabolic niches within the bacterial community of the gut, and how these activities contribute to the digestive capabilities of their host [1]. Remarkably, little attention has been focused on the structure and make up of the phage communities co-enhabiting these digestive tracts, or the potential roles that these viruses play in regulating the population density/diversity of the rumenant microbiome. A recently published study by Berg Miller et al. addresses this issue, where the authors utilzed a metagenomics based approach to assess the viral diversity within the gut of Holstein dairy cows. In the study, the authors generated a rumen-specific bacteriophage virome, which they then compared to previously published rumen microbiome data to determine if a potenital evolutionary relationship existed between phage prevelance and bacterial population abundance. Major findings of the study are described below 2. Methodology & Major Findings Methodology The authors randomly chose three Holstein dairy cows that were fed the same diet for analyzing the bacteriophage diversity within their rumens. Each subject fit into one of three catagories: actively lactating, dry (no longer lactating for offspring), or culled (no longer actively breeding). They then filter-purified phage from rumen fluid collected from each cow and purified the viral DNA in from each sample. The phage DNA was then fragmented via sonication before being randomly sequenced using pyrosequencing 2. Estimating viral poplulation diversity To estimate the viral population structure and diversity within each sample, the authors utilized the free online software, PHACCS (Phage Communities from Contig Spectrum) after first normalizing and aligning sequencing read into contigs via the Genome Relative Abundance and Average Size (GAAS) method on the CAMERA 2.0 website 3. Surprisingly little viral diversity was detected between each sample; the virome of the culled cow shared 97.5% of viral genotypes with that of the lactating cow,and 99% of the genotypes with the dry cow. In contrast, the viromes of the lactating and dry cows shared only 78.9% of genotypes 2 Viral abundance & predicted bacterial hosts The authors also classified the types and relative abundances of each virus within the cow rumen using the MEGAN metagenome analysis software to compare the virome reads to the non-redundant viral (NRV) GenBank database.Consistant with other studies 4 only 2% of the genomes were found to share significant sequence homology with known viral sequences listed in the database. Of the viral genomes that aligned with sequences within the NRV database, 36% were prediced to belong to the Siphovirdae ''family, while 28% aligned with members of the ''Myovirdae, ''and 14% within the ''Podovirdae. ''Interestingly, the predicted hosts of these viral families are primarily the ''Firmicutes, Proteobacteria, ''and ''Bacteroidetes, ''which are also the most abundant groups within rumenants. Of the viral genomes with matches in the NRV database, 68% were believed to originate from ''Firmicute hosts, while 18% were thought to target P''roteobacteria'' hosts, and 8% were thought to have originated from Bacteriodetes. Comparing CRISPR sequences of the rumen microbiome to the rumen virome Since CRISPR sequences (Clustered Regularly Interspaced Palendromic Regions) can be viewed as a form of aquired bacterial immunity against phage infection, the authors utilized NCBI's TBLASTX software to align their viral genomes with rumen microbiome data collected from previous studies to determine if an evolutionary relationship between phage infection and the structure of the rumen microbiome 5. Interestingly, the authors found that the most abundant species populating the bovine rumen encode CRISPR sequences protecting againt attack by the most prevelant phage. Summary In this study, the authors were able to identify over 28,000 different viral genotypes within the rumen of Holstein dairy cows. Of these, over 78% did not match any previously described virus. Additionally, the authors were able to determine that there was very little diversity between the viromes of Holsteins that were actively lactating, dry (no longer lactating for young), or culled (no longer breeding). More interestingly however, the majority of the identified viruses were found to target the three most abundant bacterial phyla in the rumen microbiome (Firmicutes, Proteobacteria, and Bacteroidetes), and that the most abundant species of bacteria encode CRISPR sequences for immunity against attack by these phages 2. Together, this suggests an evolutionary relationship where phages in the rumen are believed to curtail the population of unwanted species while helping to ensure that only a select few are able to proliferate. References 1 Rohwer, F., Prangishvili, D., and Lindell, D. (2009) Roles of viruses in the environment. Environ Microbiol 11: 2771–2774. 2 Berg Miller ME, Yeoman CJ, Chia N, Tringe SG, Angly FE, Edwards RA, Flint HJ, Lamed R, Bayer EA, White BA. Phage-bacteria relationships and CRISPR elements revealed by a metagenomic survey of the rumen microbiome. Environ Microbiol. 2012 Jan;14(1):207-27. 3 Angly F, Rodriguez-Brito B, Bangor D, McNairnie P, Breitbart M, Salamon P, Felts B, Nulton J, Mahaffy J, Rohwer F. PHACCS, an online tool for estimating the structure and diversity of uncultured viral communities using metagenomic information. BMC Bioinformatics. 2005 Mar 2;6:41. 4 Parsley, L.C., Consuegra, E.J., Thomas, S.J., Bhavsar, J., Land, A.M., Bhuiyan, N.N., et al. (2010) Census of the viral metagenome within an activated sludge microbial assemblage. Appl Environ Microbiol 76: 2673–2677. 5 Marraffini, L.A., and Sontheimer, E.J. 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